The NF-xcexaB family of transcription factors are involved in the regulation of a wide variety of cellular responses. These transcription factors mediate extracellular signals that induce expression of genes which are involved in such diverse processes as cell division, inflammation, and apoptosis. See, for example, Baldwin, Annu. Rev. Immunol. 12, 141-179 (1996); Beg and Baltimore, Science 274, 782-274 (1996); Gilmore et al., Oncogene 13, 1267-1378 (1996); Mayo, et al, Science 278, 1812-1815 (1997); and Van Antwerp et al., Science 274, 787-789 (1996).
NF-xcexaB is anchored in the cytoplasm of most non-stimulated cells by a non-covalent interaction with one of several inhibitory proteins known as IxcexaBs. See for example, Baeuerle and Baltimore, Science 242, 540-546 (1988). Cellular stimuli associated with immune and inflammatory responses, for example inflammatory cytokines such as tumor necrosis factor xcex1 (TNFxcex1) or interleukin-1 (IL-1), activate NF-xcexaB by inducing the phosphorylation of IxcexaBs on specific serine residues. Phosphorylation marks the IxcexaBs for ubiquitination and proteosome mediated degradation. The disruption, or dissociation, of IxcexaBs from NF-xcexaB unmasks the NF-xcexaB nuclear localization signal, and facilitates the nuclear translocation of active NF-xcexaB to the nucleus, thereby upregulating NF-xcexaB responsive target genes. See, for example, Baeuerle and Henkel, Annu. Rev. Immunol., 12, 141-179 (1994); Baldwin, Annu.Rev. Immunol., 14, 649-683 (1996); Siebenlist et al., Annu.Rev.Cell Biol. 12, 405-455 (1994); and Verma et al, Genes Dev., 9, 2723-2735 (1995). Thus, this phosphorylation of IxcexaBs is a key regulatory step for NF-xcexaB mediated processes.
Phosphorylation of IxcexaBs on two amino proximal serine residues (for example, in the case of IxcexaBxcex1 serines 32 and 36) has long been appreciated to be the major regulatory step in NF-xcexaB activation. See, for example, Baldwin, Annu.Rev. Immunol., 14, 649-683 (1996), Brown et al., Science 267, 1485-1488 (1995); DiDonato et al., Mol. Cell Biol. 16, 1295-1304 (1996); Traenckner et al., EMBO J., 14, 2876-2883 (1995). As such, an important key to elucidating the mechanism of NF-xcexaB activation, and gaining control of the immune and inflammatory responses mediated by NF-xcexaB activation, is determining the kinases involved.
Therefore, there is a need for finding kinases that are involved in the regulation of these processes. Initial attempts to identify the responsible kinase(s) revealed a specific IxcexaB-kinase activity in a large, around 700 kDa, cytoplasmic complex. Chen et al., Genes Dev. 9, 1586-1597 (1995). The activation of this kinase can be mediated by mitogen-activated protein kinase kinase kinase-1 (MEK-1), although the precise mechanism has not yet been established. Lee et al., Cell 88, 213-222 (1997).
Further experiments to decipher the functional connection between TRAFs (TNF-receptor-associated factors) and NF-xcexaB activation led to the isolation of NF-xcexaB-inducing kinase (NIK). Lee et al., Cell 88, 213-222 (1997); and Malinin et al., Nature 385, 540-544 (1997). NIK is a serine/threonine kinase which shares homology to MEKK-1. Phosphorylation of IxcexaB in response to TNFxcex1 requires NIK function. Lee et al., Cell 88, 213-222 (1997); and Malinin et al., Nature 385, 540-544 (1997); Song et al. Proc. Natl. Acad. Sci 94, 9792-9796 (1997). However, NIK does not directly phosphorylate NF-xcexaB. Lee et al., Cell 88, 213-222 (1997).
Of critical importance for elucidating, and controlling, the signaling pathways that lead to NF-xcexaB activation is the determination and characterization of kinases that directly phosphorylate IxcexaB. The abbreviation xe2x80x9cIKKxe2x80x9d is used to designate an IxcexaB kinase. Recently, an IxcexaB kinase (IKK), designated IKKxcex1, was identified in a yeast-two-hybrid screen with NIK as bait. Regnier et al., Cell 90, 373-383 (1997). IKKxcex1 was also purified using conventional biochemical techniques and determined to be the major IxcexaB kinase activity induced by TNF stimulation of HeLa cells. DiDonato et al., Nature 388, 548-554 (1997). IKKxcex1 had been cloned previously in a reverse transcriptase polymerase chain reaction (RT-PCR) based search for myc-like genes containing helix-loop-helix domains and was termed CHUK (conserved helix-loop-helix ubiquitous kinase). Connelly and Marcu, Cellular and Molecular Biology Research 41, 537-549 (1995). CHUK was renamed IKKxcex1 when its function was discovered. Regnier et al. (1997). The identification of IKKxcex1 (CHUK) as a cytoplasmic kinase which phosphorylates IxcexaB family members at their physiologically relevant sites and targets them for proteosome-mediated degradation was a major breakthrough.
The IKKxcex1 (CHUK) gene encodes a 745 amino-acid polypeptide (having a molecular mass of approximately 85 kDa). Murine and human IKKxcex1 (CHUK) cDNA clones were found to be almost identical. Connelly and Marcu, Cellular and Molecular Biology Research 41, 537-549 (1995). Another kinase, termed IKKxcex2, homologous to IKKxcex1, has also been reported. Stancovski and Baltimore, Cell 91, 299-302 (1997); Woronicz et al., Science 278, 866-869 (1997); and Zandi et al. Cell 91, 243-252 (1997). IKKxcex1 and IKKxcex2 have 52% overall similarity to each other and 65% identity in the kinase domain. Zandi et al., Cell 91, 243-252 (1997). IKKxcex1 and IKKxcex2 share two carboxy-proximal structural domains, leucine zipper and H-L-H. (Connelly and Marcu, 1995). Since these domains are thought to play roles in proteinxe2x80x94protein interactions, the IKKs may employ these domains to recruit proteins involved in their regulation or to facilitate binding to specific substrates. Recent experiments on the regulation of IKKxcex2 activation suggest that the probable interaction of the carboxy-proximal H-L-H and amino-proximal catalytic domains are required for its cytokine induced activation. (Delhase et al., 1999). An IxcexaB kinase termed T2K has been described in U.S. Pat. No. 5,776,717 to Cao.
The known IxcexaB protein kinases generally phosphorylate IxcexaBs at specific serine residues. For example, they specifically phosphorylate serines 32 and 36 of IxcexaBxcex1. Phosphorylation of both sites is required to efficiently target IxcexaBxcex1 for destruction in vivo. Moreover, activation of IKKxcex1 and IKKxcex2 occurs in response to NF-xcexaB activating agents and mutant IKKxcex1 and IKKxcex2 that are catalytically inactive block NF-xcexaB stimulation by cytokines. These results highlight the important role played by IxcexaB protein kinases in NF-xcexaB activation processes. See Stancovski and Baltimore, Cell 91, 299-302 (1997) for a recent discussion of IxcexaB kinases.
IKKxcex1 (CHUK) and IKKxcex2 have structural motifs characteristic of the IKKs. This includes an amino terminal serine-threonine kinase domain separated from a carboxyl proximal helix-loop-helix (H-L-H) domain by a leucine zipper-like amphipathic xcex1-helix structure. These structural characteristics are unlike other kinases and the domains are thought to be involved in proteinxe2x80x94protein interactions. The IKKs may employ these domains to recruit proteins involved in their regulation or to facilitate binding to specific substrates. Recent experiments on the regulation of IKKxcex2 activation suggest that the probable interaction of the H-L-H and the kinase domains are required for its cytokine-induced activation (Delhase et al., 1999).
The discovery of IKKs will facilitate elucidation of the events triggered by the engagement of cytokine receptors which lead to the activation of the cytoplasmically anchored NF-xcexaB transcription factors. This is of great importance because NF-xcexaB gene regulation is involved in a host of pathological events, in addition to inflammatory processes. For example, NF-xcexaB gene regulation has been implicated in the progression of acquired immune deficiency syndrome (AIDS), acute phase response, activation of immune and endothelial cells during toxic shock, allograft rejection, and radiation responses. Knowledge of the mechanisms of NF-xcexaB activation will be invaluable in the development of therapeutic agents for these conditions.
Significantly, the discovery of kinases that are involved in activating NF-xcexaB by phosphorylating IxcexaBs is critical for developing means for controlling cellular processes regulated by NF-xcexaB. In particular, there is a need for inhibitors of IxcexaB phosphorylation that can be used to control undesirable inflammation and immune responses. Protein kinases that act at the key regulatory step of NF-xcexaB activation provide targets for the development of inhibitors of such responses. Discovery of additional kinases involved in the phosphorylation of IxcexaBs would aid in the rational development of means for controlling cellular processes regulated by the NF-xcexaB system. Thus, there is a need for the identification and characterizing of kinases that phosphorylate IxcexaB.
An IxcexaB protein kinase which has a kinase domain, a leucine zipper like xcex1-helix domain and no helix-loop-helix has been identified (IKKxcex1-xcex94H) (U.S. Ser. No. 09/160,483). IKKxcex1-xcex94H is useful as a target for the development of inhibitors of IxcexaB phosphorylation and anti-inflammatory therapeutics.
However, a better target would be an IxcexaB protein kinase which further lacks the leucine zipper like xcex1-helix domain. Drugs that inhibit such a target would be specifically directed to the protein""s kinase catalytic domain without other interacting factors thereby providing a higher specificity of action.
The object of the present invention is to discover new IKK molecules.
These and other objects, as will be apparent to those having ordinary skill in the art, have been met by providing an isolated IxcexaB protein kinase that has a kinase domain and has neither a leucine zipper like xcex1-helix domain nor a helix-loop-helix domain (IKKxcex1-xcex94Cm). A preferred embodiment of the invention is an isolated protein having the amino acid sequence set forth in SEQ ID NO:1. Also included in this invention is an isolated IxcexaB protein kinase that contains a unique twenty amino acid sequence at the carboxy-terminal end of IKKxcex1-xcex94Cm, designated as IKKxcex1-xcex94LH, and has the amino acid sequence set forth in SEQ ID NO:4. Also included in this invention are isolated nucleic acid molecules that encode the IxcexaB protein kinase that have a kinase domain and have neither a leucine zipper like xcex1-helix domain nor a helix-loop-helix domain. (SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:6). Methods of making IKKxcex1-xcex94LH and IKKxcex1-xcex94Cm by expressing nucleic acid molecules encoding the protein are also provided. Antibodies directed to IKKxcex1-xcex94LH and IKKxcex1-xcex94Cm are also included in the invention.
The invention also includes a method of screening for an agent which modulates IxcexaB phosphorylation by the IxcexaB kinase that has a kinase domain and has neither a leucine zipper like xcex1-helix domain nor a helix-loop-helix domain, the method comprising the steps of:
incubating a mixture comprising:
the IxcexaB kinase, and
a candidate modulating agent;
detecting an agent-biased phosphorylation level of the IxcexaB kinase in the presence of the, a candidate modulating agent;
detecting an agent-independent phosphorylation level of the IxcexaB kinase in the absence of the candidate modulating agent;
comparing the agent-biased phosphorylation level with the agent-independent phosphorylation level;
selecting the candidate modulating agent that exhibits a significant difference between the agent-biased phosphorylation level and the agent-independent phosphorylation level.